Salmonella is a genus of the family Enterobacteriaceae, characterized as Gram-negative, facultatively anaerobic, non spore-forming, rod-shaped bacteria, and most strains are motile by flagella. Salmonella has an average genome GC content of 50-52%, which is similar to those of Escherichia coli and Shigella. The genus Salmonella is a pathogenic microorganism that causes infections in livestock as well as in human. Salmonella enterica, a species of Salmonella bacterium, has a variety of serovars including Gallinarum, Pullorum, Typhimurium, Enteritidis, Typhi, Choleraesuis, and derby (Bopp C A, Brenner F W, Wells J G, Strokebine N A. Escherichia, Shigella, Salmonella. In Murry P R, Baron E J, et al., eds Manual of clinical Microbiology. 7th ed., Washington D.C. American Society for Microbiology 1999; 467-74; Ryan K J. Ray C G (editors), 2004, Sherris Medical Microbiology (4th ed). McGraw Hill. ISBN 0-8385-8529-9). Of them, Salmonella Gallinarum and Pullorum are fowl-adapted pathogens, Salmonella Typhi is a human-adapted pathogen, Salmonella Choleraesuis and derby are swine-adapted pathogens, and Salmonella Enteritis and Typhimurium are pathogenic for both human and animals. Each serovar causes illness in that species, resulting in serious negative effects for farmers or consumers.
A disease of domestic birds caused by Salmonella bacterium is Fowl Typhoid (FT), which is caused by a pathogen, Salmonella Gallinarum (hereinbelow, designated as SG). Fowl Typhoid (FT) is a septicemic disease of domestic birds such as chicken and turkey, and the course may be acute or chronic with high mortality. Recently, it has been reported that Fowl Typhoid frequently occurs in Europe, South America, Africa, and South-East Asia, and damages are increasing. Outbreaks of FT in Korea have been reported since 1992 and economic losses from FT in brown, egg-laying chickens are very serious (Kwon Yong-Kook. 2000 annual report on avian diseases. Information publication by National Veterinary Research & Quarantine Service. March, 2001; Kim Ae-Ran et al., The prevalence of pullorum disease-fowl typhoid in grandparent stock and parent stock in Korea, 2003, Korean J Vet Res, 2006, 46(4): 347-353).
Pullorum disease is also caused by one of Salmonella bacteria, Salmonella Pullorum (hereinbelow, designated as SP). Pullorum disease occurs in any age or season, but young chickens are particularly susceptible to the disease. During the past century, Pullorum disease as an egg-transmitted infection has seriously affected young chickens at 1-2 weeks of age or younger in the world and Korea. In the 1980's, disease occurrence greatly decreased. However, incidence began increasing in the mid 1990's (Kwon Yong-Kook. 2000 annual report on avian diseases. Information publication by National Veterinary Research & Quarantine Service. March, 2001; Kim Ae-Ran et al., The prevalence of pullorum disease-fowl typhoid in grandparent stock and parent stock in Korea, 2003, Korean J Vet Res, 2006, 46(4): 347-353).
In Korea, outbreaks of Fowl Typhoid and Pullorum disease have been increasing since the 1990's, inflicting economic damages on farmers. For this reason, a live attenuated SG vaccine has been used in broilers for the prevention of Fowl Typhoid from 2004 (Kim Ae-Ran et al., The prevalence of pullorum disease-fowl typhoid in grandparent stock and parent stock in Korea, 2003, Korean J Vet Res, 2006, 46(4): 347-353), even though its efficacy is doubtful, and the live vaccine is not allowed to be used for layers because of the risk of egg-transmitted infections. Unfortunately, there are still no commercially available preventive strategies against Pullorum disease, unlike Fowl Typhoid. Thus, there is an urgent need for new ways to prevent Fowl Typhoid and Pullorum disease.
Meanwhile, Salmonella Typhimurium (hereinbelow, designated as ST) is a zoonotic pathogen which shows no host specificity, unlike SG or SP (Zoobises Report; United Kingdom 2003).
ST is a cause of salmonellosis in poultry, pigs, and cattle, etc. Salmonellosis is caused by Salmonella bacteria, an acute or chronic infection of the digestive tract in livestock, and shows the major symptoms of fever, enteritis, and septicemia, occasionally pneumonia, arthritis, abortion, and mastitis. Salmonellosis occurs worldwide, and most frequently during the summer months (T. R. Callaway et al., J. Anim. Sci. 86: E163-E172, 2008). In cattle, typical symptoms include loss of appetite, fever, dark brown diarrhea or bloody mucous stool. Acute infection in calves leads to rapid death, and infection during pregnancy leads to fetal death due to septicemia, resulting in premature abortion. In pigs, salmonellosis is characterized clinically by three major syndromes—acute septicemia, acute enteritis, and chronic enteritis. Acute septicemia occurs in 2-4 month-old piglets, and death usually occurs within 2-4 days after onset of symptoms. Acute enteritis occurs during the fattening period, and is accompanied by diarrhea, high fever, pneumonia, and nervous signs. Discoloration of the skin may occur in some severe cases. Chronic enteritis is accompanied by continuing diarrhea.
Once an outbreak of salmonellosis occurs in poultry, pigs, and cattle, it is difficult to halt with therapeutic agents. The reasons are that Salmonella bacteria exhibit a strong resistance to various drugs and live in cells being impermeable to antibiotics. Up to now, there have been no methods for effectively treating salmonellosis caused by ST, including antibiotics.
In addition to livestock, ST causes infections in human via livestock products, leading to Salmonella food poisoning. Consumption of infected, improperly cooked livestock products (e.g., meat products, poultry products, eggs and by-products) is a cause of human infection. Salmonella food poisoning in human usually involves the prompt onset of headache, fever, abdominal pain, diarrhea, nausea, and vomiting. The symptoms commonly appear within 6-72 hours after the ingestion of the organism, and may persist for as long as 4-7 days or even longer (NSW+HEALTH. 2008 Jan. 14).
According to a report by the CDC (The Centers for Disease Control and Prevention, USA), 16% of human food poisoning outbreaks between 2005 and 2008 attributed to Salmonella bacteria, and 18% of them were ST (Salmonella Typhimurium). With respect to Salmonella food poisoning in human between 1973 and 1984, the implicated food vehicles of transmission were reportedly chicken (5%), beef (19%), pork (7%), dairy products (6%), and turkey (9%). In 1974-1984, the bacterial contamination test on broilers during the slaughter process showed 35% or more of Salmonella incidence. In 1983, Salmonella was isolated in 50.6% of chicken, 68.8% of turkey, 60% of goose, 11.6% of pork, and 1.5% of beef. Further, a survey carried out in 2007 reported that salmonella was found in 5.5% of raw poultry meat and 1.1% of raw pork. In particular, it was revealed that ST commonly originated from contaminated pork, poultry meat, and beef (Centers for Disease Control and Prevention, CDC). A risk assessment conducted by FAO and WHO in 2002 noted that the human incidence of salmonellosis transmitted through eggs and poultry meat appeared to have a linear relationship to the observed Salmonella prevalence in poultry. This implies that, by reducing the prevalence of Salmonella in poultry, the incidence of salmonellosis in humans will correspondingly fall (Salmonella control at the source; World Health Organization. International Food Safety Authorities Network (INFOSAN) Information Note No. 03/2007). Recently, fears about food safety have been spurred by outbreaks of Salmonella from products as varied as peanuts, spinach, tomatoes, pistachios, peppers and, most recently, cookie dough (Jane Black and Ed O'Keefe. Overhaul of Food Safety Rules in the Works. Washington Post Staff Writers Wednesday, Jul. 8, 2009).
For these reasons, Salmonella infections must be reported in Germany (§6 and §7 of the German law on infectious disease prevention, Infektionsschutzgesetz). Between 1990 and 2005 the number of officially recorded cases decreased from approximately 200,000 cases to approximately 50,000. It is estimated that every fifth person in Germany is a carrier of Salmonella. In the USA, there are approximately 40,000 cases of Salmonella infection reported each year.
Therefore, there is an urgent need to control ST, which causes salmonellosis in livestock and human. The collaborative efforts of USDA and FDA have led to the development of a number of effective strategies to prevent salmonellosis that causes over 1 million cases of foodborne illness in the United States. In Denmark, conservative estimates from a cost benefit analysis comparing Salmonella control costs in the production sector with the overall public health costs of salmonellosis suggest that Salmonella control measures saved the Danish society US$ 14.1 million in the year 2001 (Salmonella control at the source. World Health Organization. International Food Safety Authorities Network (INFOSAN) Information Note No. 03/2007).
Meanwhile, a bacteriophage is a specialized type of virus that only infects and destroys bacteria, and can self-replicate only inside a host bacteria. A bacteriophage consists of genetic material being single or double-stranded DNA or RNA surrounded by a protein shell. There are three basic structural forms of bacteriophage: an icosahedral (twenty-sided) head with a tail, an icosahedral head without a tail, and a filamentous form. Bacteriophages are classified based on their morphological structure and genetic material. Based on their tail structure, bacteriophages having an icosahedral head and double-stranded, linear DNA as their genetic material are divided into three families: Myoviridae, Siphoviridae, and Podoviridae, which are characterized by contractile, long noncontractile, and short noncontractile tails, respectively. Bacteriophages having an icosahedral head without a tail and RNA or DNA as their genetic material are divided based on their head shape and components, and the presence of a shell. Filamentous bacteriophages having DNA as their genetic material are divided based on their size, shape, shell, and filament components (H. W. Ackermann. Frequency of morphological phage descriptions in the year 2000; Arch Virol, 2001, 146: 843-857; Elizabeth Kutter et al. Bacteriophages biology and application; CRC press).
During infection, a bacteriophage attaches to a bacterium and inserts its genetic material into the cell. After this a bacteriophage follows one of two life cycles, lytic or lysogenic. Lytic bacteriophages take over the machinery of the cell to make phage components. They then destroy or lyse the cell, releasing new phage particles. Lysogenic bacteriophages incorporate their nucleic acid into the chromosome of the host cell and replicate with it as a unit without destroying the cell. Under certain conditions, lysogenic phages can be induced to follow a lytic cycle (Elizabeth Kutter et al. Bacteriophages biology and application. CRC press).
After the discovery of bacteriophages, a great deal of faith was initially placed in their use for infectious-disease therapy. However, when broad spectrum antibiotics came into common use, bacteriophages were seen as unnecessary because of having a specific target spectrum. Nevertheless, the misuse and overuse of antibiotics resulted in rising concerns about antibiotic resistance and harmful effects of residual antibiotics in foods (Cislo, M et al. Bacteriophage treatment of suppurative skin infections. Arch Immunol. Ther. Exp. 1987, 2: 175-183; Kim sung-hun et al., Bacteriophage; New Alternative Antibiotics. Biological Research Information Center, BRIC). In particular, antimicrobial growth promoters (AGPs), added to animal feed to enhance growth, is known to induce antibiotic resistance, and therefore, a ban on the use of AGPs has been recently introduced. In the European Union, the use of all antimicrobial growth promoters (AGPs) was banned from 2006. Korea has banned the use of some AGPs from 2009, and is considering restrictions on the use of all AGPs by 2013-2015.
These growing concerns about the use of antibiotics have led to a resurgence of interest in bacteriophage as an alternative to antibiotics. 7 bacteriophages for control of E. coli O157:H are disclosed in U.S. Pat. No. 6,485,902 (applied in 2002-Use of bacteriophages for control of Escherichia coli O157). 2 bacteriophages for control of various microorganisms are disclosed in U.S. Pat. No. 6,942,858 (applied by Nymox in 2005). Many companies have been actively trying to develop various products using bacteriophages. EBI food system (Europe) developed a food additive for preventing food poisoning caused by Listeria monocytogenes, named Listex-P100, which is the first bacteriophage product approved by the US FDA. A phage-based product, LMP-102 was also developed as a food additive against Listeria monocytogenes, approved as GRAS (Generally regarded as safe). In 2007, a phage-based wash produced by OmniLytics was developed to prevent E. coli O157 contamination of beef during slaughter, approved by USDA's Food Safety and Inspection Service (FSIS). In Europe, Clostridium sporogenes phage NCIMB 30008 and Clostridium tyrobutiricum phage NCIMB 30008 were registered as a feed preservative against Clostridium contamination of feed in 2003 and 2005, respectively. Such studies show that research into bacteriophages for use as antibiotics against zoonotic pathogens in livestock products is presently ongoing.
However, most of the phage biocontrol studies have focused on the control of E. coli, Listeria, and Clostridium. Salmonella is also a zoonotic pathogen, and damages due to this pathogen are significant. As mentioned above, since ST has a multiple drug resistance, nationwide antimicrobial resistance surveillance has been conducted in Korea under an Enforcement Decree of the Act on the Prevention of Contagious Disease (Executive Order 16961), an Enforcement ordinance of the Act on the Prevention of Contagious Disease (Ministry of Health and Welfare's Order 179), and Organization of the National Institute of Health (Executive Order 17164). Accordingly, there is a need for the development of bacteriophages to control Salmonella. 